Registration Dossier

Diss Factsheets

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD guideline No. 439 and under GLP.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Pentaerythritol, ethoxylated, esters with acrylic acid
EC Number:
EC Name:
Pentaerythritol, ethoxylated, esters with acrylic acid
Cas Number:
Molecular formula:
(C2 H4 O)n (C2 H4 O)n (C2 H4 O)n (C2 H4 O)n C17 H20 O8
Pentaerythritol, ethoxylated, esters with acrylic acid (> 1 < 8.5 mol EO)
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Pentaerythritol, ethoxylated, esters with acrylic acid
- Substance type: UVCB
- Physical state: clear, yellowisch liquid
- Lot/batch No.: 0008190677
- Expiration date of the lot/batch: 21 February 2013
- Storage condition of test material: room temperature

Test animals

Details on test animals or test system and environmental conditions:
Human Skin
EPISKIN-SM (Source: SkinEthic, France, Batch No.:12-EKIN-016, Expiry date: 23 April 2012) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Test system

Details on study design:

Pre-incubation (Day [-1])
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed, with the media below them in contact with the epidermis, into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2.

Application and rinsing (Day 0)
- 50 μL of test item was added to each of three test skin units
- 50 μL PBS was added to each of the three negative control skin units
- 50 μL SDS was added to each of the three positive control skin units
- For additional control for staining effects of the test item, 50 μL of the test item was applied evenly to the epidermal surface.

The plates with the test item treated and the negative and positive control treated epidermis were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (22-23°C).

After the incubation time the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1x solution (0.9%) to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source care was taken to avoid the damage of epidermis. After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2.

MTT test after 42 hours incubation (day 2)
After the 42 hours incubation all EPISKIN-SM units except the one staining control were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The one additional control for coloured substances was transferred to wells filled with fresh assay medium. Then, all transferred EPISKIN-SM units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.

Formazan extraction (Day 2)
At the end of incubation with MTT a formazan extraction was undertaken: A disk of epidermis from each replicate was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 μL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).

The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for about three hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (Day 2)
Following the formazan extraction, 2×200 μL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer was read at 540 nm using acidified isopropanol solution blank (6×200 μL).

Results and discussion

In vitro

Irritation / corrosion parameter:
other: other: irritation in vitro
Remarks on result:
Basis: other: cell viability. Time point: 4 hours. Reversibility: no data. Remarks: Cell viability with the test substance was 33% compared to the negative control and therefore irritant. (migrated information)

Applicant's summary and conclusion

Interpretation of results:
Migrated information Criteria used for interpretation of results: EU
In the in vitro skin irritation test in the EPISKIN model with Pentaerythritol, ethoxylated, esters with acrylic acid the results indicated that the test item is Irritant (I) R38 [UN GHS: Category 2].
Executive summary:

Disks of EPISKIN (three units / chemical) were treated with Pentaerythritol, ethoxylated, esters with acrylic acid and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of the epidermis on each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a % relative to negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritant to skin.

Following exposure with Pentaerythritol, ethoxylated, esters with acrylic acid, the mean treated skin value was 33% and therefore irritant. All validity criteria were within acceptable limits and therefore the study can be considered as valid. In this in vitro skin irritation test in the EPISKIN model with Pentaerythritol, ethoxylated, esters with acrylic acid the results indicated that the test item is

Irritant (I) R38 [UN GHS: Category 2].